Project Summary Lung transplant involves a period of storage (ischemia) followed by the transplant (reattachment or reperfusion) event. The resultant ischemia-reperfusion (I/R) injury, clinically known as primary graft dysfunction (PGD), is a major cause of post-transplant failure. We have previously shown that lung storage induces the expression of several moieties that can ?predispose? the graft (newly transplanted lung) to inflammation and subsequent injury. Among these is the NOD like receptor protein 3 (NLRP3) inflammasome, a well characterized platform of a NLRP3 subunit and adaptor molecules whose expression and assembly are driving factors in inflammation induced cells death in a number of pathologies. Our preliminary data showed that (mouse) lung I/R increased NLRP3 expression and activity and that NLRP3 blockade reduced I/R injury. Additionally, post-transplant (human) recipients with detectable NLRP3 protein in plasma developed PGD. This implied that NLRP3 protein could be a potential risk factor for PGD. Yet the mechanism(s) by which this inflammasome is activated with lung I/R and drives injury is not known. Lung I/R differs from I/R in systemic organs in that I/R in the lung does not reflect anoxia/hypoxia-deoxygenation effects alone; rather it also represents signaling associated with ?sensing' stop and restart of flow. Lung I/R, as our earlier investigations show, initiates NADPH oxidase 2 (NOX2) activation and reactive oxygen species (ROS) generation, followed by activation of transcription factors NF?B and AP-1. We also reported an increase in intracellular calcium [via voltage gated calcium channels (VGCC)]. Our hypothesis is that NOX2 activates the NLRP3 inflammasome; once activated this inflammasome is a major driver of I/R injury (i.e. PGD). Our goal is to employ models, tools, techniques and information from our past work on lung I/R signaling to ascertain if NLRP3 is regulated by NOX2 (Aim 1), and/or by rise in intracellular calcium via various sources including VGCC (Aim 2). Finally we will determine the mechanism by which activated NLRP3 inflammasome drives PGD (Aim 3). For this application, the lung I/R models used will comprise of in vitro (pulmonary microvascular endothelial cells in flow chambers), in situ (isolated murine and human lungs), in vivo (hilar cross clamp) and mouse lung transplant models as well as human plasma banked at the Lung Transplant Outcomes group (LTOG). These models will be used on NOX2 null, cell type specific NOX2 null, VGCC null and NLRP3 reporter mice to evaluate the mechanism of NLRP3 regulation and its role in I/R injury. The multidisciplinary team of investigators to pursue these aims includes 2 human ex vivo donor lung experts, an epidemiologist with expertise in lung injury, a mouse transplant group, and the PI, a lung biologist who first identified a link between NOX2 and onset of inflammation with lung storage and transplant. Expertise on neutrophil biology, and on the NLRP3 inflammasome will be provided by consultants.